The purpose of this project is to develop multicompartment neuronal cell microculture chambers in a 96 well format to produce high throughput (HTP) in vitro neurocircuitry models using human neuronal cells. This platform allows HTP phenotyping of neurons derived from induced pluripotent stem (iPS) cell lines carrying risk gene alleles associated with susceptibility towards substance abuse. In particular, this work will aid the understanding of synaptic plasticity changes associated with the pathogenic behaviors of drugs of abuse, including addiction to cocaine, nicotine and alcohol. Synaptic plasticity changes, especially in the mesolimbic system, are associated with addictive behavior and are not recapitulated with existing cell culture systems using human neurons. We have constructed a multicompartment, microfluidic system that allows us to model the neuronal circuitry of the mesolimbic reward system. However, given the increasing number of iPS cell lines available, the need to include genome edited versions of each genotype with isogenic genetic background, the multiple subtypes of neurons required, as well as the need for HTP screening for small molecule compounds that can reverse the alterations associated with the gene variants, the current analysis is difficult or impossible to scale up to meet with the need fo HTP analysis of defined neurocircuitry. Thus, in this CEBRA application, we propose to build upon our previous success in creating neurocircuitry models by constructing a microfabricated HTP platform, compatible with the GE IN CELL high-content imaging system, for both morphological and functional (Ca2+-imaging) analyses. Following construction of the HTP system, we will validate it by focusing on a well-studied genetic variant associated with increased alcohol consumption, nicotine and cocaine abuse, containing an altered ?-opioid receptor (MOR) sequence that alters intracellular signaling. The Single Nucleotide Polymorphism (SNP) rs1799971 produces a non-synonymous amino acid substitution in MOR (OPRM1 A118G), replacing Asparagine 40 (MOR N40) with a variant with Aspartate (MOR D40). We have prepared multiple iPS cell lines carrying either homozygous MOR N40 or MOR D40 from subjects with well-characterized alcohol abuse, nicotine dependence-related behaviors, and known ethnicity. Our goal in this exploratory project is to develop a novel HTP experimental paradigm to support in vitro mini- neurocircuits mimicking the midbrain DA neurocircuitry that is formed by neurons generated from patient specific iPS cells carrying defined risk gene variants, to allow the screening of small molecule compounds to reduce phenotypes consistent with addictive behaviors.